The present invention relates to a thin-layer chromatography apparatus according to the generic term of claim 1.
Thin-layer chromatography is a widespread process for the separation and analysis of substance mixtures. There, a stationary phase is provided as a thin layer on a suitable carrier made of glass, polyester, aluminum or the like. The substance mixture to be investigated is applied at a starting point or a starting line on the stationary phase. The thin-layer plate is then developed. For this a mobile phase is brought as eluant (Eluens) onto the starting line or the starting point, say by placing the thin-layer plate in a trough chamber from where the mobile phase begins-to run, especially by the capillary action of the stationary phase on the thin-layer. The mobile phase there can carry along over the thin-layer plate constituents of the substance mixture to be investigated and that are soluble in that phase. The less-well-soluble components remain on the stationary phase. If the individual components of the applied substance mixture, by reason of substance-specific retention values, show different distributions between mobile and stationary phase, this leads to a breaking-up of the substance mixture over the thin-layer plate. There is obtained a spatial distribution of the components over the thin-layer plate that is substance-specific. The analysis of the distribution pattern, therefore of a chromatogram, makes possible qualitative and quantitative statements about the components of the substance mixture.
The required analysis of the developed thin-layer chromatography plates occurs ordinarily optically with thin-layer chromatography apparatuses, in which the thin-layer plate is irradiated with light, and with which it is possible to investigate how the irradiated light is altered by the thin-layer plate.
The present invention concerns itself more precisely with such an optical thin-layer chromatography apparatus, and it is not relevant whether the application and development proper of the thin-layer plate also occur in the apparatus itself; further, it is insignificant whether the carrier is rigid or flexibly formed from glass, polyester or the like, so long as it is accessible to an optical investigation of the developed chromatogram.
The optical determination as to which substances are present in the chromatogram can be supported, on the one hand, by the place which a certain component of the substance mixture has reached on the thin-layer plate in the development, and, on the other hand, by its optical properties, i.e. the absorption capacity of the substance, its fluorescent properties, etc. An exact investigation requires, therefore, both a high local resolution and also an exact spectral investigation.
It has been proposed (xe2x80x9cDevelopment of a system for measuring the UV spectra of components separated by TLCxe2x80x9d by S. Ebel and W. Windmann in J. Planar Chromatography, 1991, pp. 171 ff), to conduct light from a deuterium lamp over a fiber-optic bundle onto a thin-layer plate and to feed it from there to a photo diode array detector. The fiber-optic bundle consists of a xe2x80x9cYxe2x80x9d fiber which is used to conduct the light of the deuterium lamp onto the thin-layer plate, and to conduct it from there to the optical examination arrangement.
Measures with such systems are described in a large number of publications, for example A. N. Diaz and F. Garcia Sanchez in xe2x80x9cFiber optic fluorescence scanning in thin-layer chromatographyxe2x80x9d in xe2x80x9cInstrumentation Science and Technology, 22 (3), 273-281 (1994)xe2x80x9d; A. N. Diaz xe2x80x9cFiber-optic remote sensor for in situ fluorimetric quantification in thin-layer chromatographyxe2x80x9d in Analytica Chimica Acta, 255 (1991) 297-303.
A further device is known from xe2x80x9cDetermination of Reflectance of Pesticide Spots on Thin-Layer chromatograms Using Fiber Opticaxe2x80x9d by M. Beroza, K. R. Hill and K. H. Norris in Analytical Chemistry, vol. 40, 1608-1613 (1968). There it is proposed to scan a thin-layer plate with stochastically arranged Y-light conductors. The foot of the Y-fibers is led to the thin-layer plate, its first arm to a light source and its other arm to an optical investigation device. A reading head is required for the plate scanning and a head for the reference measurement on an adjacent free path.
In the article xe2x80x9cA versatile Spectrophotometric Scanner for Large TLC Platesxe2x80x9d by B. L. Hamman and M. L. Martin in Analytical biochemistry vol. 15, 305-312 (1966) it is proposed to install in a light-proof housing a plate scanner which comprises a driven slide piece, with which a fiber bundle is moved over a thin-layer plate. Sending and receiving photo-conductors are arranged facing one another lying opposite on the slide piece. The light spot size can be determined by the sliding of slots of suitable size in front of the sending photo-conductor.
A problem with such thin-layer chromatography apparatuses lies in the uneven distribution of the substances over the regions to which they are carried on the thin-layer plate during the development. The concentration is highest in the center of a zone and decreases toward the edges of the spot.
A known possibility for avoiding such faults lies in illuminating a large spot. In a further known arrangement (S. Bayerbach and G. Gaulitz xe2x80x9cSpectral detection in thin-layer chromatography by linear photo diode array spectrometryxe2x80x9d in Fresenius Z. Anal. Chem. (1989) 335:370-374) there is proposed, therefore a light spot size of one to two millimeters dependent on the distance from the thin-layer plate. This takes place also in use of unordered fiber light bundles.
It was further proposed (S. Ebel, E. Geitz and J. Hocke in xe2x80x9cFully automatic, computer-controlled evaluation of thin-layer chromatogramsxe2x80x9d in GIT Fachz. Lab, 24th year, pp. 660 ff) to lead the light spot in meander pattern over the substance spots on the thin-layer plate. It has further been proposed to deflect the thin-layer plate by a definite amount over an eccenter?, in order to avoid the point-form measuring of a thin-layer chromatogram spot, cf. vol. 5 xe2x80x9cDxc3xcnnshicht-chromatographiexe2x80x9d of Ullmans encyclopedia of technical chemistry.
What is disadvantageous with the previously described measurements by means of which the uneven substance distribution over the spot is supposed to be compensated for, is regularly the reduced local resolution. It leads with a given amount of substance to a worsened separating performance, or with limited amounts of substance to worsened detection sensitivity.
From WO 93/14392 there is known an analysis device with a polychromatic light source. The light source can comprise a glow lamp light source with a small opening or, preferably, a semiconductor light source, for example an LED.
From EP 0 060 709 B1 there is known a process and a device for multidimensional real-time chromatography. The prior known system has first means for the chromatographic separating of a sample of an unknown composition into a first grouping of components, in which these first means can be a matter of a chromatographic thin-layer plate; second means for the further separating of the components mentioned into a higher-dimensional second group of components; third means for the recognizing of the components mentioned. The third means can be a linear multi-photodiode array which detects how a light source illuminates the eluting solvent on the thin-layer plate. The illumination can use a plurality of light sources which are used successively and emit at different wavelengths that are specific for the unknown components sought. The publication concerns itself also with the problem that the analysis is not very accurate because of the spreading of the spots. The known process proposes the real-time investigation during the separation, but allows there in particular no subsequent evaluation of already developed thin-layer plates. It is therewith not possible to draw upon the prior-known system for the investigation of filed thin-layer plates, as required, say, for the documentation of charge qualities in the pharmaceutical industry.
The dissertation xe2x80x9cRemission spectra: Possibilities and limitsxe2x80x9d von W. Windmann (University of Wxc3xcrzburg, 1991) concerns itself with the use of light conductors in thin-layer chromatography apparatuses. It is stated (page 144) that it is absolutely necessary to achieve high intensities of the irradiated light on the thin-layer plate in order to obtain a sufficiently strong remission signal, that, on the other hand however, the spot size must be sufficiently large so that a spot on its path is still perceived if the chromatographic development has faults. The use of larger fiber bundles (allegedly) can solve these problems only within narrow limits.
A device in which a large number of coherent parallel light beams is used in order to determine the position of an object is known from EP 0 669 515 A3. The known device is used in order to determine the position of tree trunks or other long stretched-out objects in a channel or the like.
The present invention aims to make new information available for industrial use and, in particular, but not exclusively to make ready a sturdy, compact thin-layer chromatography apparatus with high spectral and local resolution, in which also in particular in any case slight disturbances arise through ambient light and which is nevertheless economical to manufacture.
The solution of the aim set is claimed independently; Preferred forms of execution are found in the dependent claims.
A basic thought of the invention lies therefore in arranging the light conductors in a predetermined geometric alignment.
Therewith there is obtained first of all a regular, preferably uniform illumination or detection over the spot width by a plurality of light conductors. It is thus possible directly to achieve a desired light intensity distribution over the entire substance spot. The alteration of the irradiated light by absorption and/or fluorescence can then likewise be detected over the entire spot width. The sliding-in of blinds for the achievement of a desired illumination and scanning geometry is not absolutely required.
The geometric arrangement of the sending and receiving light conductors to one another makes it possible purposefully to influence the overlapping of the coverage of the light spot from a sending-light conductor with the coverage that is detected by the receiving-light conductors and to make it controllable according to wish. The regularity of the arrangement which is conditioned by the fact that sending and receiving light conductors are also geometrically arranged among one another, provides that the overlapping integrals of different sending and receiving conductors allocated to one another correspond, which holds also if for certain purposes not all the overlapping integrals are exactly identical to one another, but groups of corresponding overlapping integrals are present, say in the case of use of several different types of fibers. The local resolution is determined in the proposed arrangement by the comparatively small overlapping integral and not by the spot size of the entire illuminated thin-layer plate zone.
Coverage fields of the receiving-light conductors and light spots of the sending-light conductors can adjustably partially overlap, it being possible to adjust the distance of the light conductors from the thin-layer plate, this being possibly done by piezoelectric modulation or with mechanical means.
Preferably at least tenxe2x80x94generally however clearly morexe2x80x94sending-light conductors are provided, which are arranged linearly closely adjacent to one another. The light spots of in each case adjacent sending-light conductors on the thin-layer plate can nearly overlap or be slightly spaced from one another. In this case a receiving-light conductor preferably receives light from at least two sending-light conductors.
With two or more parallel series of sending-light conductors next to one another a given plate is more intensely illuminated and by lighting from different sides the angular distribution of the received light becomes more uniform. If light of different spectral intensity is irradiated in each case into different light conductors there is yielded on the thin-layer plate a desired, say nearly uniform illumination spectrum simply by arrangement of the particular light conductors. The illumination with parallel light conductors makes it possible moreover to irradiate light perpendicularly onto the plate and to collect the light remitted perpendicularly to the plate for evaluation. This is advantageous according to the Lambert cosine law for the achievement of high light intensities.
The series are movable with respect to one another for the adjustment of the light-spot overlapping of the sending-light conductors of different series, say by swing and/or translation of one series toward the other or away from this.
An adjustably variable spacing of the sending-light conductors within a series makes possible measurements with different overlappings of the light spots with one another or with particular coverages and therewith different local resolutions.
Input-side-coupling of light from different light sources into the sending-light conductors facilitates the obtaining of a desired illumination spectrum. An intensity approximately uniform over the spectral range used ensures that the detectors used in the optical investigation apparatus used are not overloaded by especially intense signals in narrow spectral ranges. A desired illumination spectrum is possible to achieve also with a single light source by means of dispersive elements between light source and thin-layer plate, or by light conductors of different length.
A refractive lens as dispersive element improves, moreover, the coupling efficiency into the sending-light conductors. The coupling efficiency is also increased if a possibly present fiber mantle on the lamp side is removed, possibly detached away, and the ends of several fibers are possibly welded or cast in synthetic resin. For an especially uniform illumination of the thin-layer plate with at least nearly the same intensity over all the sending light conductors, between light source and input side of the sending light conductors there is arranged a diffusing screen or the like.
While it is advantageous per se to provide a plurality of optical-sending light conductors, preferably also or at least a plurality of optical reception light conductors is provided, such as at least twenty, preferably, however, between 50 and 100 receiving light conductors.
The plurality of optical receiving- light conductors can be arranged for simultaneous reception of light transmitted through the thin-layer plate and of light remitted from the thin-layer plate or from the substances presented on the thin-layer plate. Also if light is simultaneously received, in each case optical investigation arrangements of their own are not absolutely necessary. Economically, the receiving-light conductors provided for the remission/fluorescence and transmission are led to one and the same optical investigation device and evaluated there in alternation. For this the light path between optical investigation apparatus and thin-layer plate can be partitioned off in selective alternation, say by means of a chopper, or the light conductors are coupled in alternation into the optical investigation apparatus. Therewith there takes place a quasi-simultaneous evaluation.
An especially preferred variant of the thin-layer chromatography apparatus has at least one series of sending-light conductors and one series (row) of receiving-light conductors, preferably with light conductors arranged tightly each over 10 thick, next to one another.
If only a single row of sending-light conductors and receiving light conductors is formed, sending-light conductors and receiving-light conductors can be arranged in common on Y-fibers ending on the thin-layer chromatography plate or in alternation in one row. While with the use of Y-fibers a 100% overlapping of sending-light spots and receiving viewing fields is given, this will not be the case with sending- and receiving-light conductors arranged in one row, which is advantageous if through a smaller overlapping integral an increased local resolution is sought. The overlapping integral can be influenced especially by the spacing between light conductor ends and the thin-layer plate.
When sending-light conductors radiate light of different spectral intensity, each receiving light conductor receives light. This improves the optical evaluation by the investigation apparatus.
The sending- and receiving-light conductors can be arranged in different parallel rows, in which case an offset arrangement xe2x80x9con the gapxe2x80x9d (auf Lxc3xcckexe2x80x9d) between sending- and receiving-light conductor row is preferred, in order to achieve a better light yield. An equal diameter for all the sending- and receiving-light conductors is not absolutely necessary there, which, if need be, makes possible a tighter packing.
Also different materials are usable, say quartz fiber for the detection of ultraviolet light, and teflon fibers for the range above 250 nm.
If so desired, the light-conductor arrangement can be arranged into a broad broom, in which 30 fibers each with a 100 xcexc diameter yield a broom width of about 3.5 mm, and 50 fibers yield already 5.5 mm. On the edges a shading can be performed, if this is too much for a given thin-layer plate, say because of only small amounts of material. For this at will the coupling of light into the outer sending-light conductors can be suppressed at the light source, the radiation at the plate, radiation of light into the receiving-light conductors and/or the coupling of light from the receiving-light conductors into the optical investigation apparatus.
Sending- and receiving-light conductors are preferably moved with a step motor or the like relatively to the thin-layer plate, in order to scan a chromatography path. Preferably in the case of a rigid scanning head the plate is moved. This avoids changes of the light transmission in the light conductors through movement-conditioned refraction index variations, say on bending.
The orientation of the thin-layer plate is visually documented with a CCD camera or-the like.
Also a means can be provided for the detection and/or for the compensation of non-plane-parallel lying thin-layer plates. For the determination of the plate alignment, the distance of the sending and/or scanning head from the plate, at points of the plate not affected by solvent and substance, can be determined with reference measurements as white standard. Such points are to be found, for example, behind the eluting running end of the outer paths, or to the side of the outer paths. The reference measurement can determine, say, the entire light intensity in reflection. If the intensity of the reflected light on the different separate points is not equal, then the plate does not lie parallel. A previously executed measurement can then be rejected or a measurement still being carried out is suppressed. What is important above all is an oblique position which simulates (or fakes) differences from path to path, since typically the analysis occurs by comparison with substances running on adjacent paths. An oblique position leading to differences within a path practically causes no trouble with such measurements, but can nevertheless easily be balanced out. The sending- and or receiving- light conductors in the present arrangement for the obtaining of small sending light spots and therewith of high local resolution can thereby be led close to the plate; ordinarily distances are clearly under one millimeter, such as a few 100 xcexc. The correction prevents the situation that through oblique positions such as could be caused by grains of sand or the like under the thin-layer plate, measurement errors could arise.